Growing cells in a reservoir formed of a flexible sterile plastic liner

ABSTRACT

A method and apparatus for aseptic biological production of cells and/or microorganisms are provided. The apparatus includes a support housing having an interior chamber, a disposable flexible sterile plastic liner lining the interior chamber and a head plate attached to the liner forming a sealed chamber with the liner to form a reservoir. After use the liner can be thrown away and the apparatus can be reused with a new liner. In this way, the apparatus simplifies cleaning and ensuring validation required by pharmaceutical and food industry standards. Aerating fluid is introduced during culturing of cell or microorganisms in the reservoir. A growth characteristic can be used to vary flow rate and/or composition of the aerating fluid. Another reservoir may be used for circulation between two reservoirs.

PRIORITY APPLICATIONS

This application is a divisional of application Ser. No. 09/388,239filed on Sep. 1, 1999, now U.S. Pat. No. 6,245,555 B1. The presentapplication also claims priority to U.S. Provisional Application No.60/098,701 filed Sep. 1, 1998, No. 60/125,656 filed Mar. 22, 1999. Eachof the foregoing applications are hereby incorporated herewith as iffully set forth herein.

FIELD OF THE INVENTION

The present invention relates to the field of biologic cell production.More specifically, the present invention relates to the asepticproduction and processing of cells and/or microorganisms in abioreactor.

BACKGROUND

The production of chemicals in bioreactor systems is expensive. Thedifferential between production costs and product market value is thedominant driving force for drug discovery and development of potentialbioprocesses. The importance of production costs is reflected in theeconomic observation that the volumetric productivity of a wide range ofbiologically produced products is about the same at $0.17 per liter perday. The expense of biological production is a motivation for pursuingchemical synthesis when possible; however, the complexity of synthesisof many natural products often makes this route equally costly. In theabsence of chemical synthesis, metabolites derived from microorganismsmust be produced in aseptic bioreactors. In contrast, plant-derivedchemicals can be harvested from intact plants. Therefore, agronomicproduction or collection from natural environments is a formidablecompetitor to growth of plant tissues in bioreactor systems. Manyrationales are given for pursuing plant tissue culture as a potentialproduction system. The most compelling are those situations where intactplants are poor competitors. Some plants either grow very slowly, or arenot amenable to agronomic production. In addition, environmentaldegradation is limiting the attractiveness of natural harvest,particularly from endangered environments such as the rain forest wherethe biochemical diversity is the greatest.

Although the bioreactor described herein is not limited to use for planttissue culture, the economic constraints and stringent asepsisrequirements presented by this production system provide an excellentcontext to demonstrate the effectiveness of the bioreactor. There havebeen many efforts to commercialize plant metabolites from cell culture;however, few have achieved commercial success. Low productivity isusually cited as the reason for failure despite the fact that productionrates and tissue concentrations are very often substantially higher thanthe intact plant. In fact, tremendous productivities have been achievedby plant tissue culture. There are at least eight different systemswhere the metabolite levels are greater than 10% of the cell dry weight,and several of these productivities have been achieved with culturesthat display relatively high growth rates. One example is anthocyaninpigments production by P.C.C. Technology, Japan where the cell contentwas greater than 17% and effective specific growth rates were 0.22 day⁻¹at a scale of 500 L. Rosmarinic acid production was successfully scaledup by A Nattermann & Cie GmbH in a 30 L stirred tank. The titer ofrosmarinic acid reached 5.5 g/L with volumetric productivity of nearly 1g/L/day, and tissue content as high as 21% of dry weight. The failure ofthese processes is more a failure to compete economically with wholeplant material rather than a failure of the cultures to be biochemicallyproductive. There have also been significant advances in strategies toimprove cellular productivity by cell line selection, geneticengineering, elicitation and root culture or enhance reactorproductivity by operational strategies such as high density culture,integrated product recovery and immobilization. However, there is alimit to the improvements that can be achieved by these strategies, andfor compounds where there is a low-cost alternative from intact plantmaterial, it simply does not make sense to attempt production inbioreactor systems.

Despite the limitations, the potential of plant-tissue culture derivedchemicals has resulted in a tremendous investment from both industry andacademia in developing this technology. It has been demonstrated thatlarge-scale production is technically feasible. The first commercialprocess was the production of shikonin. Since the market for this dye islimited, production has been on hold to focus efforts on taxol as a moreprofitable target. Similarly, the efforts of EscaGenetics on theproduction of vanillin were way-layed in favor of taxol development.Ginseng has been produced commercially by Nitto Denko (Japan) for 10years at a scale of 25,000 liters. There are other reports of industrialscale cultivation of plant cells including tobacco at 15,500 L and threedifferent plant species by DIVERSA (Hamburg, Germany) up to 75,000 L.Taxus sp. is being grown at industrial scale by both Phyton, Inc. andSam Yang. These examples show that technical problems of scale-up can beovercome.

The preceding indicates that the technology for production of chemicalsby plant tissue culture is available provided the secondary metabolitehas a sufficiently high value. The required product price to considerplant tissue culture production has been estimated to be in the range of$1000 to $5000 per Kg. The issue arises as to whether this technologycan be extended to lower value/higher volume biochemical production. Toachieve this objective, it is useful to understand what contributes toproduction costs.

Based on experience with the commercial development of shikonin, theMitsui group estimated that 64% of the production costs for culturedplant cells was due to fixed costs (depreciation, interest and capitalexpenditures). A similar number can be calculated from the recentanalysis presented by Goldstein based on general plant tissue culturecharacteristics. Using Goldstein's 2000 kg product per year basis (whichis implicitly 22 tons of cell mass based on assumed productivity), thefixed costs (calculated as capital charges) were 55.4% of themanufacturing costs. The estimate of Yoshioka and Fujita is likely to bemore generally applicable since it uses a cycle time of roughly 14 daysas compared to the 5-day reactor cycle time assumed by Goldstein.Clearly capital investment is an important target for cost reduction.This is not surprising since equipment and support facilities associatedwith aseptic bioprocessing are extremely expensive because vessels areconstructed of stainless steel and pressure rated for autoclavesterilization. Accordingly, eliminating the need for expensive autoclaveconstruction could substantially reduce production costs by reducing theinitial capital investment.

SUMMARY OF THE INVENTION

In light of the foregoing, the present invention provides a method andapparatus for producing cells, tissues and/or microorganisms. The methodincludes providing a disposable liner forming a reservoir having anopening. A closure is attached to the liner to close the opening. Theliner and attached closure are sterilized. A biomass dispersion is thenintroduced into the reservoir.

The present invention further provides a bioreactor for culturing cells,tissues and microorganisms. The bioreactor includes a support, and aliner mounted on the support and forming a reservoir for receiving abiomass dispersion. A closure sealingly engages the liner to close theliner opening. The closure sealingly engages the liner and is separablefrom the liner. The closure includes an inlet port in fluidcommunication with the reservoir.

DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe preferred embodiments, will be better understood when read inconjunction with the accompanying drawing, in which:

FIG. 1 is a side sectional view of an aseptic bioreactor in accordancewith the present invention.

FIG. 2a is a graph of the growth of the cell cultures for Examples 1 and2; and

FIG. 2b is a graph of the growth of the cell culture for Example 3.

FIG. 3 is a side sectional view of second embodiment of an asepticbioreactor in accordance with the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Referring now to the drawings in general, and to FIG. 1 specifically, anaseptic bioreactor 10 is illustrated. The bioreactor is lined with adisposable liner 30. The bioreactor is used for biological production ofcells, tissues and/or microorganisms in a culture medium. After use, thecells and culture medium are removed from the bioreactor 10 and theliner 30 is disposed. The bioreactor can then be used again with a freshliner.

The bioreactor 10 can be used to produce any of a number ofmicroorganisms and/or cells, including, but not limited to bacteria,fungi, animal cells, nematodes and plant cells. In addition to theproduction of biomass, the bioreactor is operable to process biomassgrown in alternative bioreactors or tissues produced by conventionalnon-aseptic methods, such as: animal husbandry, field growth plants orgeneral biomass byproducts. Specifically, the bioreactor is operable inconnection with biotransformation of biochemicals utilizing enzymaticcapabilities of tissue-such as transgenic plant tissue grown in thefield to express a heterologous enzymatic activity. The bioreactor isalso operable in connection with expressing proteins from post-harvestactivated promoters in which the biomass is grown in the field. In suchcircumstances, the tissue is not growing within the vessel, however, theviable tissue is carrying out the desired chemistry within a processvessel that can be of the type described herein.

In the following description, the bioreactor is described in connectionwith the production of plant cells. However, the design and/or operationof the bioreactor can be modified to accommodate production of othertypes of cells. For example, the production of plant cells progressesslowly so that respiratory heat removal is not problematic to maintainthe proper temperature within the bioreactor. However, when producingmicroorganisms that exhibit rapid progression, such as bacteria,respiratory heat removal is generally necessary to maintain the propertemperature within the bioreactor. Therefore, to accommodate such anapplication, the bioreactor can be configured to include a heat exchangecoil for heat removal. Accordingly, although the following descriptionexemplifies use of the bioreactor in connection with producing plantcells, the bioreactor is not limited to such use.

The bioreactor 10 includes a hollow support housing 20 lined by thedisposable liner 30. A head plate 40 is attached to the liner to form asealed reservoir within the liner 30. The head plate 40 and liner 30 arethen sterilized to form an aseptic environment within the reservoir.Culture medium is introduced into the container 20 through aninoculation tube 46 that is in fluid communication with the reservoir.The culture medium is then inoculated through the inoculation tube 46.An aerator 50 extends through the head plate 40 to aerate the biomassdispersion during the production cycle. During the production cycle, thereservoir is sealed to prevent contamination from the outsideenvironment. After the production cycle is complete, the biomassdispersion is removed from the reservoir. After the biomass dispersionis removed, the liner 30 is disposed. Since the liner 30 prevents thebiomass dispersion from coming into contact with the support housing 20,the support housing need not be sterilized before reusing the bioreactorfor another production cycle. Instead, the reusable head plate 40 isattached to a new liner, and the two are sterilized. In this way, thesterilization of the bioreactor is simplified, and validation of the“clean in place” procedures is significantly simplified.

STRUCTURE OF THE APPARATUS

The structure of the bioreactor 10 will now be described in greaterdetail. The support housing 20 is an elongated hollow generallycylindrical container. The upper end of the support housing is generallyopen and the lower end or bottom of the support housing is closed. Inthe present instance, the support housing 20 includes a circumferentialflange 24 projecting outwardly from the side walls 22 of the housing.Baffles 26 are disposed in the interior of the support housing 20adjacent the bottom on the support housing. As shown in FIG. 1, thebaffles 26 form an asymmetric or offset V-shape. As discussed in moredetail below, the V-shape improves circulation of the bioreactor.

The support housing 20 can be configured in almost any shape. If an“air-lift” circulation system is utilized as described below,preferably, the aspect ratio of the container is such that the height is2 to 5 times the width of the container. However, the aspect ratio maybe higher or lower. For instance, in certain situations it may bedesirable to use a support housing that is shallow and wide. For such ashallow support housing, the fluid pressure of the aeration fluid can bereduced because the pressure at the bottom of a shallow reservoir isless than that of a deeper reservoir, and the aeration fluid mustovercome this fluid pressure to aerate the reservoir.

The support housing 20 may be formed of glass thereby exposing thebiomass dispersion 15 to light, and allowing visual inspection of thebiomass dispersion. However, the support housing can also be formed ofany number of materials including metal or plastic. In addition, thesupport housing can be opaque or semi-opaque.

As can be seen from the foregoing, the configuration of the supporthousing 20 can be varied considerably. Generally, a structure thatmechanically supports the weight of the cell suspension 15 can beutilized. Specifically, a structure that supports the vertical force ofthe weight of the cell suspension 15 and the lateral or horizontal forceof the fluid pressure of the biomass dispersion can be utilized. Forinstance, the support housing 20 could be in the form of an openframework or mesh rather that the solid walls of the housing shown inFIG. 1.

The liner 30 is preferably formed of plastic. The plastic may betransparent or translucent to allow light into the reservoir and topermit visual inspection of the bioproduction/bioprocessing if desired.The type and thickness of plastic will depend upon several variables,including the size of the support housing 20 and the type ofsterilization process that will be utilized to sterilized the liner 30and the head plate 40. For instance, a 2 mil thickness autoclavablepolypropylene bag can be utilized if autoclaving is used. For othersterilization processes, such as gas-phase or plasma-phasesterilization, the liner 30 may be formed of a 6 mil thicknesspolyethylene bag.

A shown in FIG. 1, the area in the support housing 20 above the baffles26 and between the side walls 22 forms an internal chamber. The liner 30lines this internal chamber to prevent the cell suspension 15 fromcoming into contact with the interior of the support housing 20. Theupper edge of the liner 30 overlays the flange 24 at the top of thesupport housing 20. The upper edge of the liner 30 may be sandwicheddirectly between the head plate 40 and the flange 24. However, in thepresent instance, the upper edge of the liner 30 is disposed between thehead plate and a support ring 28 that rests on the flange 24. Thesupport ring 28 is a flat ring that is approximately as wide as theflange 26 of the support housing. By sandwiching the upper edge of theliner 30 between the head plate 40 and the support ring 28, the headplate and liner can be removed from the support housing withoutdetaching the liner from the head plate.

The head plate 40 is a substantially round plate having a diameter thatis greater than the open end of the support housing 20. The head platecan be made from a variety of materials including metal and plastic. Forinstance, the head plate 40 may be formed of polycarbonate. The lowersurface of the head plate 40 confronts the upper surface of the supportring 28. Preferably, a sealing groove 42 extends around the periphery ofthe lower surface of the head plate, spaced inwardly from the outer edgeof the plate. A seal 44 in the form of an O-ring is disposed in thesealing groove 42. The head plate 40 can be attached to the sealing ring28 in any of a number of ways. Preferably the head plate is releasablyattached to the sealing ring 28 so that the liner 30 can be detachedfrom the head plate, allowing the head plate to be reused. In thepresent instance, the head plate 40 is clamped to the sealing ring 28with the upper edge of the liner 30 disposed between the head plate andthe sealing ring. In this way, a fluid-tight seal is provided betweenthe head plate and the opening in the liner.

The bioreactor 10 includes an inoculation tube 46 and a sampling tube 48that project into the sealed reservoir formed by the liner 30 and thehead plate 40. The inoculation tube 46 and the sampling tube 48 are influid communication with the sealed reservoir and provide access to theinterior of the bioreactor from exterior of the bioreactor. A seal isformed between the exterior of the inoculum and sampling tubes 46, 48and the head plate to provide a fluid-tight seal. The inoculation tube46 is of sufficient diameter for introducing the culture medium into thesealed reservoir through the inoculation tube, along with the inoculum.Depending on the application, the inoculation tube can be configured inany one of a number of designs that facilitate introducing inoculum andresealing the inoculation tube to prevent contamination. The samplingtube is of sufficient diameter for withdrawing media samples.

The bioreactor 10 further includes an aerator 50. In the presentinstance, the aerator is a sparger. A filter is provided for filteringthe gas used by the aerator to prevent contaminants from entering thereservoir. As shown in FIG. 1, the aerator projects through the headplate 40 and into the bottom of the reservoir. For submerged cultures,the aerator 50 is preferably aligned with the vertex of the V-shapedbottom of the reservoir formed by the baffles 26 in the bottom of thesupport housing 20. In the present instance, the aerator 50 includes twospargers attached in a T-configuration that is parallel to the crease inthe V-shaped baffle 26.

In the present instance, the aerator 50 also operates to circulate thebiomass dispersion within the bioreactor 10. This is referred to as an“air-lift reactor.” The gas bubbling through the biomass dispersion 15from the aerator 50 causes variations in the density of the culturemedium. This operates to circulate the culture medium in the reservoir.For this reason, it is desirable to have the bottom end of the aerator50 adjacent the lowest point of the reservoir.

The bottom of the reservoir may be flat. However, to improve circulationit is desirable to eliminate the lower corners that form dead spaces.For this reason, the baffle 26 is V-shaped. Offsetting the vertex of theV-shaped baffle 26 as shown in FIG. 1 further improves the circulationin the reservoir. In addition, in a large bioreactor it may be desirableto include a plurality of aerators to improve aeration and circulation.

As described above, in the present instance the aerator 50 operates asan aerator and a circulator. In certain instances it may be desirable tofurther include a separate circulator. For instance, an impeller can beincluded to improve the circulation in the reservoir. Such an impellercan be journalled in a bearing mounted in the head plate so that itextends downwardly into the bottom of the reservoir.

As another example, a magnetic impeller may be provided to improve thecirculation in the reservoir. A magnetic impeller would not require themechanical connection between the impeller blade and the drivemechanism. The magnetic impeller utilizes magnetic force to drive theimpeller blade. Since there is no mechanical connection between themagnetic impeller blade and the impeller drive, there is no need toprovide an additional seal as is required around the drive shaft of astandard impeller. In addition, since magnetic impeller blades can berelatively inexpensive, the magnetic impeller blade can be disposedalong with the liner 30 after a production cycle is completed. Thisfurther simplifies cleaning the apparatus and preparing for a subsequentproduction cycle.

The embodiment illustrated in FIG. 1 shows a gas-dispersed liquidculture. However, the process can also be implemented as aliquid-dispersed bioreactor in which medium is dispersed over the top ofthe biomass. For example, referring to FIG. 3, an alternative bioreactorembodiment 110 is illustrated. In the alternative embodiment, thebiomass is in a primary reservoir and fluid is circulated into theprimary reservoir from a secondary reservoir 180 that is separate fromthe primary reservoir.

Specifically, the bioreactor 110 includes a reservoir lined by adisposable liner 130 that lines a support 120. A head plate 140 sealsthe liner as in the first embodiment and forms a primary reservoir forgrowing or processing a biomass. Preferably, a bed of material 123, suchas industrial process packing is introduced into the primary reservoirand culture medium is circulated through the reservoir.

A secondary reservoir 180 is also provided. Preferably, the secondaryreservoir is also formed by a disposable liner that lines a support andis sealed by a head plate. The secondary reservoir contains a quantityof fluid, which may be culture medium or biomass dispersion as isdiscussed further below.

A feed line 163 and return line 164 are provided for recirculating fluidbetween the primary and secondary reservoirs. The feed line and returnline are in fluid communication with the primary and secondaryreservoirs. The return line 164 extends into the bottom of the primaryreservoir to recycle fluid after it flows through the primary reservoir.

In this way, a “trickle bed” can be utilized to grow or process biomass.Specifically, biomass is introduced into the primary reservoir. Culturemedium is circulated through the primary reservoir from the secondaryreservoir. If a bed of material 123 is provided, the culture mediumflows into the primary reservoir over the bed of material 123. Theculture medium trickles through the bed of material and is then recycledthrough the return line 164. As the fluid flows through the primaryreservoir over the bed of material 123, the fluid promotes the growth orprocessing of the biomass in the bed of material.

Depending on the biomass, the circulated fluid can be either culturemedium alone or biomass dispersion. Specifically, if the biomassdispersion is a suspension, then the suspension is circulated from thesecondary reservoir, into the primary reservoir, over the bed ofmaterial, and then recycled through the return line 164. In otherapplications, the biomass dispersion may not be a suspension. In suchapplications, substantially all of the biomass remains in the primaryreservoir and culture medium is circulated over the biomass.

The re-circulating system has been described in connection with a bed ofindustrial process packing through which the culture medium or biomassdispersion circulates to promote growth or processing of biomass in thebed. However, the bedding material need not be industrial processpacking. The bedding material can be other granular material or matrixmaterial.

In addition, the recirculating system is operable in certainapplications without a bed of material. In such applications, thebiomass is introduced into the primary reservoir, and culture medium iscirculated over biomass.

METHOD OF OPERATION

The bioreactor 10 operates as follows. The head plate 40 and liner 30are attached and sterilized to form an aseptic container. The head plateand liner can be sterilized in any of a number of ways. For instance,autoclaving can be used if the liner 30 and head plate are formed ofmaterials that withstand the temperature and pressure of the autoclavingprocess. Alternatively, the head plate and liner can be sterilized bygas-phase sterilization using ethylene oxide gas in accordance withhospital guidelines for ethylene oxide sterilization of medical devices.In addition, other sterilization processes such as a vapor phaseoxidant, plasma or radiation sterilization.

Furthermore, although the operation of the device contemplates theability to be sterilized in accordance with the stringent guidelines ofthe pharmaceutical industry, the term sterilization as used herein isnot limited to pharmaceutical or medical sterility. The termsterilization is meant to include cleaning to less stringent standardssuch as the standards for sterility for the food industry. In addition,as used in this description, the term sterilization is also meant toinclude marginal sterility, meaning reduction or suppression ofnon-productive contaminating organisms to sufficiently low numbers sothat their presence does not prevent or significantly impede the desiredbiomass growth or processing. Accordingly, as can be seen from theforegoing, operation of the bioreactor is not limited to a particularsterilization process.

If sterilization is to be used, the head plate 40 and attached liner 30can be inserted into the autoclave without the support housing becausethe support housing need not be sterilized since it does not come intocontact with the biomass dispersion. Accordingly, a liner and head platecan be autoclaved in a collapsed state, so that a smaller autoclavevessel can be utilized to service a plurality of large-scale productionbioreactor tanks. If gas-phase sterilization is to be used, the headplate is attached to the liner, a gas mixture of ethylene oxide andcarbon dioxide is introduced into the sealed reservoir formed by thehead plate and liner. Preferably, prior to introducing a gas into thereservoir, steam is introduced into the reservoir to provide a dampenvironment and assure hydration of contaminant spores. After gas-phasesterilization, the toxic ethylene oxide is removed by aeration using aslow flow of air. Surface sterilant gases such as hydrogen peroxide mayrequire little or no aeration.

During gas-phase sterilization, the ethylene oxide gas can permeate theliner 30 and accumulate between the liner and the inner wall of thesupport housing 20. This accumulated ethylene oxide may remain in thebioreactor after standard aeration procedures. This residual ethyleneoxide can permeate through the liner 30 into the reservoir during theproduction cycle to adversely affect cell production. Accordingly, it isdesirable to minimize the gap between the liner and the inner wall ofthe support housing. In addition, it is desirable to aerate the insideand outside of the liner and head plate assembly to reduce the amount ofresidual ethylene oxide. This can be accomplished by removing the sealedhead plate and attached liner assembly from the support housing andaerating the support housing in addition to aerating the reservoir. Itshould be noted that potential toxicity of residual ethylene oxiderelease will become less significant for larger scale reactors since thefilm surface area to tank volume rapidly declines with scale-up. Inaddition, the use of non-permeating surface sterilant gas could greatlyreduce aeration considerations for gas or plasma phase sterilization.

After the liner and head plate assembly are sterilized, the liner isinserted into the support housing. Alternatively, depending on the typeof sterilization used, the liner and head plate assembly can be insertedinto the support and then sterilized. Culture medium is then introducedinto the sealed medium through the inoculation tube 46. If desired, agrowth regulator is also introduced into the reservoir through theinoculation tube. This combination is then inoculated with a cellculture. For clarity, the resultant mixture of cell culture, growthregulator, if any, and culture medium are referred herein to as abiomass dispersion. The biomass dispersion is aerated by the aerator 50during the production cycle.

The biomass growth is monitored during the production cycle so that thecomposition of the aeration fluid and/or the flow rate of the aerationfluid can be adjusted to optimize cell growth. Depending on the cell ormicroorganism being produced, the characteristics monitored during theproduction cycle may vary. For instance, utilizing dissolved oxygen as acontrol parameter may be desirable for rapid-production cultures such asbacteria. Conversely, using dissolved oxygen as a control parameter forslow-production cultures, such as plant tissue cultures, can beproblematic. Similarly, off-gas analysis can be used to monitorrespiration. However, off-gas analysis is generally more amenable tomonitoring rapid-production cultures then slow-production cultures.Nonetheless, other measurements indicative of biomass growth can beutilized to effectively monitor biomass growth of slow-productioncultures. For instance, in certain culture media, the refractive indexof the culture medium is an indicator of sugar levels, and theelectrical conductivity of the culture medium is an indicator ofinorganic nutrients. These characteristics can be measured and used tocorrelate biomass accumulation based on nutrient consumption. Theexamples detailed below describe plant tissue production, utilizingrefractive index and electrical conductivity and medium osmolality ascontrol parameters. These details of such production control are setforth in greater detail in Ramakrishnan, D.; Luyk, D.; Curtis, W.R.,“Monitoring biomass in root culture systems”, Biotechnology andBioengineering, 62 (6): 711-721, 1999, which is hereby incorporatedherein by reference as is fully set forth herein.

None of these measurements mentioned above require introduction of ameasuring instrument into the reservoir. However, if the bioreactor isutilized to produce other types of cells and/or organisms, thebioreactor may include one or more measuring devices that extend intothe reservoir, preferably through the head plate in a fluid-tightrelation, to prevent introduction of contaminant cells and/ormicroorganisms. For instance, a pH meter may be attached to the headplate, extending downwardly into the reservoir to monitor the pH of theculture medium.

In response to the measured characteristic, the operating environmentmay be modified to optimize biomass production or processing. Forinstance the gas composition of the aeration gas can be modified.Alternatively, the flow rate of the aeration gas can be varied. Inaddition, the biomass dispersion may be supplemented with sugar and/orinorganics to encourage continued growth.

EXAMPLE 1

Two 9 L (6.5 L working volume, w.v.) bioreactors were provided: onesteam autoclave sterilizable and the other ethylene oxide (EtOX)sterilizable. For the autoclavable configuration, the plastic liner wasa 2 mil polypropylene autoclavable bag (25 cm width by 46 cm height).The top edge of the bag was clamped to a 12.7 mm thickness polycarbonatehead plate which contained a 17 mm internal diameter (ID) stainlesssteel inoculation tube, a 1.4 mm ID tube for withdrawing medium samples,a 4.5 mm ID gas outlet tube, and an additional 4.5 mm ID tube whichextended to the bottom of the bag with a 0.2 micron sintered metalmobile phase sparger attached for sparging of gas. A bead of hot meltglue stick was applied between the bag and head plate, which melted uponautoclaving to provide an air-tight aseptic seal. The reactor was steamsterilized at 121° C. for 30 minutes in a collapsed state, and expandedafter autoclaving inside a 15.2 cm diameter non-sterile glass vessel.

EXAMPLE 2

The second bioreactor is designed to eliminate the need for autoclavesterilization. The second bioreactor utilizes sterilization throughexposure to ethylene oxide (EtOX)-carbon dioxide gas mixture. Theability to sterilize the reactor without high temperature eliminated theneed for autoclavable materials. For this reason, the plastic liner wasconstructed from 6 mil thickness polyethylene, and Norprene tubing wasused in all connections to minimize degradation and diffusion of EtOXduring the sterilization cycle. The liner was constructed to fit insidethe same 15.2 cm ID (internal diameter) glass column, with the seamssealed with a hot glue gun and a hand iron wrapped in paper towel. Thehead plate was constructed from 20-gauge (0.95 mm) 304 stainless steelsheet metal where the ports analogous to those described for thepolycarbonate head plate were silver-soldered in place. The head plateand liner were held in place by bolting through the collar of theCorning conical glass connections. A bead of silicone glue was appliedbetween the headplate and plastic to facilitate the seal. Gas-phasesterilization was accomplished using a commercially available mixture ofethylene oxide: 10% EtOX in carbon dioxide to avoid the danger offlammability. Prior to gas introduction, steam under ambient conditionswas briefly introduced from the release valve of a commercial pressurecooker. This pre-sterilization steam treatment provided a dampenvironment and assured hydration of contaminant spores. Exposure toethylene oxide was accomplished in a fume hood where roughly fourreactor volumes of the EtOX gas mixture were introduced three times overa period of two days. Gas was introduced through the air sterilizationfilter, and the gas outlet was rotated using pinch clamps through themedium sample, gas outlet, and inoculation ports. After gas-phasesterilization, the toxic EtOX was removed by a slow flow of 200 mL/minof sterile air for a period of roughly two days based on hospitalguidelines for ethylene oxide sterilization of medical devices.

EXAMPLE 3

A 40 L bioreactor with a working volume (w.v.) of 28.5 L was constructedfrom 6 mil plastic to fit inside a 59.7 cm height by 28.3 cm diameterglass tank. The head plate sealed against a compression ring so that theliner and head plate could be independently removed from the tank tofacilitate aeration from both sides of the liner and eliminate pocketsof ethylene oxide between the liner and the support tank. Circulationwas facilitated by off-center placement of the sparger, and contouringof the tank bottom with a baffle. The baffle was ‘V’-shaped with thecrease off-center to align with the sparger. The sparger consisted oftwo 0.2 micron sintered metal mobile phase spargers attached in aT-configuration so that they could be placed parallel to the crease inthe V-shaped baffle. The reactor was fitted with a 28.6 mm IDinoculation port and 9.5 mm ID sample port similar to the other reactorconfigurations. The 34 cm ID head plate and compression ring wereconstructed of 12.7 mm thickness polycarbonate with an o-ring groovemachined in the upper plate 25.4 mm from the edge to provide for a sealagainst the plastic liner.

EXAMPLE 4

A 150 L (100 L w.v.) bioreactor was constructed from 6 mil polyethylenefilm to fit inside a 85.7 cm height by 45.1 cm diameter stainless steelprocess tank. The baffling geometry, head plate compression ring, andsparging arrangement were analogous to Example 3. The polycarbonate headplate included a 19.1 mm ID inoculum port, and two 12.7 mm IDcompression fittings for the sparge tube inlet and sample port. Aseparate 6.4 mm ID compression fitting was used for gas outlet. A thinsilicone film on the 5.7 cm width aluminum compression flange 28 wasutilized to accomplish the seal between the head plate and plasticliner.

The root culture used in EXAMPLE 4 was culture of Hyoscyamus muticus,line HM90T, established by Agrobacterium rhizogenous transformation in1990 and grown on “Gamborg B5” medium. The cell suspension line used forthe four EXAMPLES was established from the same root culture byde-differentiating the root culture through the addition of 0.2 mg L⁻¹of the growth regulator 2,4 dichlorophenoxyacetic acid (2,4 D). The rootline and cell lines have been maintained for more than 3 years throughserial bi-weekly subculturing on their respective medium. Autoclaved B5medium was introduced aseptically for Examples 1 through 3, and Example4 utilized filter sterilization through a cartridge filter sterilizationunit. The initial medium volumes were 6.5 L (Example 1), 6.7 L (Example2), 28.5 L (Example 3) 100 L (Example 4, for both cell and root culturerun).

The operational strategy for the bioreactors relied upon the refractiveindex and conductivity (as well as visual observation through the glassreactor) for making operational changes. The initial gas flow rate tothe prototype reactors was minimized (0.05 volumes of gas per volume ofmedium per minute, VVM) corresponding to 0.33 liters per minute. Air wasused for the first day to avoid potential problems of oxidative stressand the low flow rates reduced foam fractionation and loss of cells onthe reactor vessel walls at the surface of the medium. When theconductivity started to decline indicating culture growth—the gascomposition was changed to 30-40% oxygen in air by oxygensupplementation. This oxygen enrichment permitted low gas flow rates andminimized wall growth. The gas flow rate was incrementally increased to0.25 VVM for the 9 L autoclaved bag and 0.20 VVM for the 9 L EtOXsterilized bag as the culture nutrients declined. The gassing programfor the 40 L reactor was initiated at 0.05 VVM, and incrementallyincreased to 0.25 VVM as the culture nutrients declined. CO and Osupplementation for the 40 L reactor were initiated at day 2 and 10,respectively. The glass support tank permitted visual observation, whichconfirmed that the gas flow should be increased more rapidly in theair-lift reactors than the gas-flow used in a typical stirred tank toavoid cell sedimentation.

The gassing strategy in the 150 L bioreactor paralleled that of thesmaller reactors. This pilot-scale run was undertaken to verify theutility of nutrient feed based on the measurements of mediaconductivity, refractive index and osmotic pressure. When refractiveindex and conductivity indicated 90% nutrient consumption (day 9 afterinoculation), a sucrose feed corresponding to an additional 20 g sucroseper liter was added to the bioreactor. The root culture was operated insimple batch mode without media supplementation.

RESULTS

Biomass accumulation for the two smaller vessels is plotted in FIG. 2a.Growth ceased in the EtOX sterilized reactor in Example 2 due to sugardepletion as indicated by a zero refractive index for the final cellconcentration data points. The autoclaved reactor in Example 1 alsoreached a zero RI; however, a supplementation of sugar was added at day11, which permitted continued growth. This indicated that substantiallyhigher cell concentrations are possible. The autoclaved bag reactor wasterminated when a low-level contaminant was observed at day 13 as theappearance of slow growing colonies on LB media plates.

Growth in the ethylene oxide sterilized reactor in Example 2 was clearlyattenuated—presumed to be due to toxicity of residual ethylene oxide.Based on recommendations for hospital use of EtOX, it was anticipatedthat the two days aeration period should have been adequate for removalof residual EtOX. However, it was later realized that the sand below theplastic bag could have acted as a reservoir that would diffuse smallamounts of residual EtOX for a long period of time. After 15 days ofslow growth, the rapid consumption of nutrients indicated a rapid growthperiod, which was visually evident through the glass reactor as well,suggesting a recovery from the ethylene oxide toxicity.

As discussed previously, residual sterilant gas removal predicated thatthe potential for residual toxicity can be minimized for a well fitreactor liner. For the Third Example, the contouring of the supporthousing was accomplished by baffles which resulted in large reservoirvolumes between the liner and the tank wall. The design of the 40 Lbioreactor permitted removal of the liner and head-plate from the tankto provide aeration on both sides. AS shown in FIG. 2b, thissuccessfully eliminated the long lag observed in the smaller prototypereactor in Examples 1 and 2, and the specific growth rate of 0.26 day⁻¹is approximately the same as the results for stirred tank bioreactors.The bioreactor of the Third Example produced 2.9 Kg FW (199 g DW) ofcells in 13 days.

The 150 L bioreactor with sugar supplementation produced over 53.8 Kgfresh weight (1.5 Kg DW) of plant cell suspension biomass during the 33day culture period. This represents one of the highest biomassproductivities achieved in plant cell culture at the pilot scale. Thisrun demonstrated the ease of scaleability of the bioreactor and utilityof the monitoring technique. By the end of the run, more than half ofthe water within the bioreactor was inside the biomass so that moresimplified methods of attempting to monitor bioreactor performance basedon media concentrations were not accurate. The same reactor inoculatedwith homogenized root tissue produced 24.1 g FW Kg FW (881 g DW) or roottissue for a 31 day culture period. The growth of root culturesdemonstrates the versatility of the bioreactor in growing an organizedtissue.

The method and apparatus of the present invention provide a simple, lowcost bioreactor for bench and pilot scale discovery and developmentalstudies. A plastic lined reactor has very attractive characteristicsbesides the reduction in costs. Such a design could potentiallyeliminate much of the costs associated with validation of clean in place(CIP) procedures since the plastic liner portion of the reactor isdisposable. Validation of sterilization only requires verifyingsterilization of the reusable head plate, and a clean plastic liner.Accordingly, the method and apparatus of the present invention should becapable of meeting the very stringent cGMP (good manufacturingpractices) guidelines currently imposed on the pharmaceutical industry.

Costs for gas sterilization could be improved by sterilization in acollapsed state to minimize gas use. Similarly, the bag and head plateassembly could be autoclaved in a collapsed state—thereby utilizing onesmaller autoclave-type vessel to service many larger-scale productiontanks.

It will be recognized by those skilled in the art that changes ormodifications may be made to the above-described embodiments withoutdeparting from the broad inventive concept of the present invention. Forinstance, the bioreactor may be utilized for anaerobic fermentation. Forsuch production, the bioreactor need not include means for aerating orcirculating the cell suspension in the reservoir. Accordingly, it shouldbe understood that the present invention is not limited to theparticular embodiments described herein but is intended to include allchanges and modifications that are within the scope and spirit of theinvention as set forth in the claims.

What is claimed is:
 1. A method for culturing one or more cell ormicroorganism, comprising the steps of: providing a flexible sterileplastic liner forming a reservoir; introducing a culture media into thereservoir; inoculating the culture media with one or more cell ormicroorganism to provide a cell suspension for culturing the cell ormicroorganism; aerating the cell suspension with a aerating fluid at aflow rate wherein the aerating fluid comprises a gas; detecting a growthcharacteristic of the cell or microorganism in the cell suspension; andvarying the flow rate and/or the composition of the aerating fluid inresponse to the detected characteristic.
 2. The method of claim 1comprising the step of releasing aerating fluid from the reservoir. 3.The method of claim 1 comprising the step of circulating the cellsuspension.
 4. The method of claim 1 wherein the reservoir comprises anopening and the method comprises the step of providing a closure havinga port in fluid communication with the reservoir, wherein the closureforms a fluid-tight seal with the reservoir to seal the opening.
 5. Themethod of claim 4 wherein the step of inoculating comprises inoculatingthe culture media through the port in the closure.
 6. The method ofclaim 4 wherein the step of aerating comprises aerating the cellsuspension through the port in the closure.
 7. The method of claim 1wherein the step of aerating comprises bubbling the aerating fluidthrough the cell suspension.
 8. The method of claim 4 comprising thestep of circulating the cell suspension through the port in the closure.9. The method of claim 1 comprising the step of sealing the reservoir toprevent a contaminant cell or microorganism from entering the reservoir.10. A method for culturing one or more cell or microorganism, comprisingthe steps of: providing a disposable first flexible sterilized plasticliner forming a reservoir having an opening; attaching the first linerto a closure to close the opening; introducing into the reservoir a cellsuspension comprising culture medium and one or more cells ormicroorganisms; culturing the cell or microorganism in the reservoir;detaching the first liner from the closure after culturing the cell ormicroorganism; and attaching a second flexible sterilized plastic linerto the closure after detaching the first liner.
 11. The method of claim10 comprising the step of aerating the cell suspension with a fluid at aflow rate.
 12. The method of claim 10 comprising the step of circulatingthe culture medium within the reservoir.
 13. The method of claim 11comprising the step of detecting a growth characteristic of the cell ormicroorganism in the cell suspension and varying the aerating fluidcomposition in response to the detected characteristic.
 14. The methodof claim 13 wherein the reservoir is translucent, and the step ofdetecting a growth characteristic comprises optically detecting acharacteristic of the cell suspension while the cell suspension is inthe reservoir.
 15. The method of claim 11 comprising the step ofdetecting a growth characteristic of the cell suspension and varying theaerating fluid flow rate in response to the detected characteristic. 16.The method of claim 15 wherein the reservoir is translucent, and thestep of detecting a growth characteristic comprised optically detectinga characteristic of the cell suspension while the cell suspension is inthe reservoir.
 17. A method for culturing cells and/or a microorganism,comprising the steps of: providing a first flexible sterile plasticlining forming a reservoir having a first opening: introducing a culturemedia into the first reservoir; introducing cells or a microorganisminto the first reservoir; closing the first opening so that the firstopening is substantially closed while maintaining a port for fluidtransfer into the first reservoir; providing a second reservoir having aculture media; circulating the culture media between the first reservoirand the second reservoir through the port in the first opening.
 18. Themethod of claim 17 comprising the step of circulating the culture mediawithin the second reservoir.
 19. The method of claim 17 comprising thestep of circulating the culture media within the second reservoir withan aerating fluid.
 20. The method of claim 17 comprising the step ofaerating the cells or microorganism with a fluid.
 21. The method ofclaim 20 wherein the cells or microorganism are aerated by aerating theculture media in the second reservoir.
 22. The method of claim 20wherein the culture media and the cells or microorganisms are combinedto form a cell culture, wherein the method comprises the step ofdetecting a growth characteristic of the cell or microorganism in thecell culture and varying the flow rate and/or composition of theaerating fluid.
 23. The method of claim 22 wherein the method comprisesvarying the flow rate of the aerating fluid.
 24. The method of claim 22wherein the method comprises varying a composition of the aeratingfluid.
 25. The method of claim 22 wherein one of the first and secondreservoirs are translucent and the step of detecting a characteristiccomprises optically detecting a characteristic of the cell culture whilethe cell culture is in the one of the first and second reservoirs. 26.The method of claim 17 wherein the second reservoir is formed of aflexible plastic liner having a second opening.
 27. The method of claim17 wherein the step of closing the first opening comprises releasablysealing the first opening.